Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
Embryonic stem cells, derived from the inner cell mass of mammalian blastocysts, have the capability to grow indefinitely while maintaining the ability to generate all cell and tissue types in the body (pluripotency). These properties have lead to expectations that human embryonic stem cells (hESCs) might be useful to treat patients with various diseases and injuries, thereby revolutionizing regenerative medicine.
Cell transplantation therapy using stem cells may offer a viable treatment strategy for patients with brain disease or injury, such as Parkinson's disease, Huntington's disease, stroke or spinal cord injury by providing new cells to replace those lost through disease. However, the clinical application of hESCs faces difficulties regarding ethical concerns relating to the use of embryos, as well as instances of tissue rejection after implantation due to immunological incompatibility between patient and donor cells.
One way to circumvent these issues is to artificially derive an embryonic stem cell-like (pluripotent) cell from a mature somatic cell by inducing a “forced” expression of certain genes. These artificially derived embryonic stem cell-like cells are known as induced pluripotent stem (iPS) cells and are believed to be identical to embryonic stem cells in many respects (Hochedlinger, K. & Plath, K; (2009) Development 136, 509-523). The generation of iPS cells from mature somatic cells, such as fibroblast cells obtained directly from the patient, prevents therapeutic concerns regarding ethics and/or tissue rejection, and may potentially provide the optimal cell source for regenerative medicine.
iPS cells were first generated by Yamanaka and colleagues in 2006 from mouse fibroblast cells (Cell 126, 663-676). The method of deriving iPS cells traditionally involves the transfection of certain embryonic stem cell-associated genes into non-pluripotent cells, such as mature fibroblasts. Transfection is usually achieved-through viral vectors, such as retroviruses. Yamanaka and colleagues ((2006) Cell 126, 663-676) initially identified 4 key genes essential for the production of pluripotent stem cells: Oct-3/4, Sox2, c-Myc and Klf4. Additional studies demonstrated the requirement of Nanog as a another major determinant of cellular pluripotency (Okita, K., Ichisaka, T. & Yamanaka, S. (2007) Nature 448, 313-317; Wernig, M. et al. (2007) Nature 448, 318-324; and Maherali, N. et al. (2007) Cell Stem Cell 1, 55-70). In 2007, two independent research groups generated iPS cells from human cells (Takahashi, K. et al. (2007) Cell 131, 861-872; and Yu, J. et al. (2007) Science 318, 1917-1920). Applying the same principles used earlier in mouse cells, Yamanaka and colleagues (Takahashi, K. et al. (2007) Cell 131, 861-872) successfully transformed human fibroblasts into pluripotent stem cells using the same 4 pivotal genes Oct-3/4, Sox2, c-Myc and Klf4 in a retroviral transfection system. Thomson and colleagues (Yu, J. et al. (2007) Science 318, 1917-1920) used Oct4, Sox2, Nanog and Lin28 using a lentiviral transfection system. The exclusion of c-Myc in these experiments was based on evidence that c-Myc is oncogenic and is not necessary to promote cellular pluripotency.
The use of neural precursor cells derived from hESCs or iPS cells bears great therapeutic potential for the treatment of neurological disorders and injuries such as Parkinson's disease, Huntington's disease, stroke or spinal cord injury through the generation of replacement neural cells. Currently cell transplantation therapy of neural precursor cells requires in vitro differentiation of the neural precursor cells from hESCs or iPS cells.
As reported, both hESCs and iPS cells can be efficiently differentiated into neural precursor cells, using either spontaneous or factor-induced differentiation protocols. Those neural precursor cells are capable of giving rise to neuronal and glial cells both in culture and in vivo (Wernig, M. et al. (2009) Proceeding of the National Academy of Science 105, 5856-5861; Dottori, M. & Pera, M. F. (2008) Methods Mol Biol 438, 19-30; Reubinoff, B. E. et al. (2001) Nature Biotechnology 19, 1134-1140; Reubinoff, B. E., Pera, M. F., Fong, C.-Y., Trounson, A. & Bongso, (2000) Nature Biotechnology 18, 399-404; Itsykson, P. et al. (2005) Molecular and Cellular Neuroscience 30, 24-36; Pera, M. F. et al. (2004) Journal of Cell Science 117, 1269-1280).
Previous work, including that of the inventors, demonstrates that hESC-derived or iPS-derived neural precursor cells survive transplantation into the injured adult rodent brain and differentiate towards both neuronal and glial cell fates—some studies demonstrating recovery of function (i.e: Bjorklund, Sanchez-Pernaute et al. (2002) PNAS 99: 2344-2349; Kim, Auerbach et al. (2002) Nature 418: 50-56; Ben-Hur, Idelson et al. (2004) Stem Cells 22(7): 1246-1255, Dinsmore, Ratliff et al. (1996) Cell Transplantation 5(2): 131-143; Dihne, Bernreuther et al. (2006) Stem Cells 24(6): 1458-1466; Riess, Molcanyi et al. (2007) Journal of Neurotrauma 24(1): 216-225; Song, Lee et al. (2007) Neuroscience Letters 423(1): 58-61; Aubry, Bugi et al. (2008) PNAS; Dali, Zhi-Jian et al. (2008) Stem Cells 26(1): 55-63; Hatami, Mehrjardi et al. (2009) Cytotherapy 11(5): 618-630; Hicks, Lappalainen et al. (2009) European Journal of Neuroscience 29(3): 562-574, Vazey et al. (2010) Cell Transplantation, 19; 1055-1062).
However, the formation of tumours, such as teratomas, following transplantation of hESC-derived neural precursor cells has been observed in a number studies (Roy, N. S. et al. (2006) Nat Med 12, 1259-1268; Erdo, F. et al. (2003) J Cereb Blood Flow Metab 23, 780-785 (2003); Hedlund, E. et al. (2007) Stem Cell 25, 1126-1135; Pruszak, J., Sonntag, K.-C., Aung, M. H., Sanchez-Pernaute, R. & Isacson, O. (2007) Stem Cells 25, 2257-2268; Bjorklund, L. M. et al. (2002) Proceeding of the National Academy of Science 99, 2344-2349; (Riess, Molcanyi et al. (2007) Journal of Neurotrauma 24(1): 216-225; Aubry, Bugi et al. (2008) PNAS; Vazey et al. (2010) Cell Transplantation, 19; 1055-1062)., and was noted by Wernig and colleagues (2009) Proceeding of the National Academy of Science 105, 5856-5861) in a recent study in which iPS cell-derived neural precursors were transplanted into a 6-OHDA lesion model of Parkinson's disease. The formation of teratomas is thought to result from a proportion of the transplanted cells retaining an undifferentiated (i.e. pluripotent) state. Accordingly, teratoma formation following transplantation of hESC- or iPS cell-derived neural precursor cells presents a major obstacle for the clinical application of stem cell therapy, as tumour formation as a clinical result of cell transplantation therapy in human patients is unacceptable.
In light of the limitations shown for hESCs and iPS cells (including ethical considerations, tissue rejection and tumourgenicity), a need for a source of cells for central nervous system (CNS) transplantation therapy exists.
As said above, reprogramming mature somatic cells, as demonstrated by the generation of iPS cells, removes ethical concerns raised over the use of hESCs and also allows for the transplantation of cells obtained from the patient's own body (autologous transplantation), addressing issues of tissue rejection. However, the use of iPS cells does not address the concerns of tumour formation associated in transplantation therapy resulting from co-transplantation of a proportion of non-committed pluripotent cells.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.